Diagnostic news and trends from the Colorado State University Veterinary Diagnostic LaboratoriesVolume 15, Number 1 Spring/Summer 2010

VETERINARY DIAGNOSTIC LABORATORIES

SPECIAL ISSUEINSIDE:FOCUS ONPCR

Diagnosticians use two primary methods to detect

infectious diseases: detect the organism or detect

antibodies against the organism. Most frequently, they

detect infectious agents in biological specimens by

culture, cytology, fecal examination, histopathology,

immunological techniques and nucleic acid amplifi ca-

tion techniques.

The polymerase chain reaction (PCR) assay ampli-

fi es DNA. When a reverse transcriptase step is incorpo-

rated, it can convert RNA to DNA and then amplify it

(RT-PCR). These are the most commonly used nucleic

acid amplifi cation techniques. Detection of the organ-

ism gives the most information supporting a clinical

diagnosis of an infectious disease, but these assays

aren’t available nor optimal for all agents. Thus, anti-

body detection is still commonly used to diagnose

infectious diseases. In some situations, the combina-

tion of organism detection and antibody detection

assays is indicated. Bartonella spp. infection in cats

and Ehrlichia spp. infections in dogs, for example. For

these organisms, PCR-positive results can occur prior

to seroconversion, so the PCR can be benefi cial to

prove current infection in the acute cases. In contrast,

the PCR assays can become negative later in the course

of disease as the immune responses develop; however,

serum antibodies are generally detectable at that time.

This situation may occur in many other infectious die-

seases, as well.

MANAGING FOR PREDICTABILITYWhen evaluating infectious disease diagnostic test

results, the analytical sensitivity defi nes the minimum

detectable amount of the substance in question that

can accurately be measured; the analytical specifi city

defi nes whether the substance detected cross-reacts.

The diagnostic sensitivity is the proportion of positive

test results from known infected animals; the diagnos-

tic specifi city is the proportion of negative test results

from known uninfected animals. The predictive value

of a positive test, or PPV, is the probability that a

test positive animal is diseased; the predictive value of

a negative test, or NPV, is the probability that a neg-

ative animal is normal. The lower the prevalence of

disease the lower the predictive value of a positive. Dis-

ease prevalence has little effect on negative predictive

values.

Depending on the infectious agent in question, PCR

and RT-PCR assays can be more sensitive than other

available assays. In addition, PCR assay results can

often be returned within 24 hours of sample submis-

sion, which is generally quicker than culture. However,

because special equipment is required, the assays must

be shipped to a diagnostic lab. If the organism in ques-

tion is diffi cult to culture, can’t be cultured or takes

a long time to culture, such as some

viruses, Leptospira, Ehrlichia or myco-

bacteria, PCR assays are of particular

benefi t. In contrast, some PCR assays

are less sensitive than other currently

available organism demonstration tech-

niques. For example, Giardia spp. IFA

or antigen tests are both more sensitive

than Giardia spp. PCR assays, probably

because of PCR inhibitors in feces.

Specifi city of PCR assays can be high,

depending on the primers used in the

reaction. For example, primers can be

designed to detect one pathogen genus

but not others. Primers can also be

designed to identify only one species. For

example, a PCR assay has been developed

to detect all Trichomonas spp. or just one

species, such as T. foetus.

QUALITY CONTROL IS CRITICALPCR assays are prone to false-positive

results if sample contamination occurs

during collection, transportation or test-

ing. Some PCR assay results may also be

affected by administration of anti-micro-

bial drugs prior to sample collection. For

example, haemoplasma or Bartonella spp.

Advances in Molecular Diagnostics

PCR detection of infectious diseases Michael R. Lappin, DVM, PhD, DACVIM,CSU VDL Endocrinology and Special Serology Lab Supervisor

CLINICAL TIPS

Sensitivity, specifi city, positive predictive value and negative predictive value vary with each PCR assay. Many of the infectious disease agents encountered in practice colonize normal animals as well as induce disease in some individuals. Thus, the PPV of many PCR assays can be low. Veterinarians generally need to use a combination of fi ndings to help diagnose infectious disease:■ Appropriate signalment and

history for the infectious agent suspected

■ Clinical signs referable to the agent

■ Detection of the agent (cytology, culture, antigen assay, PCR assay) or antibodies against the agent

■ Exclusion of other causes of the clinical syndrome

■ Response to an appropriate treatment

When these criteria are met, the suspected infectious agent might have been the cause of the clinical disease. However, it is always possible that the disease process resolved in spite of the therapy prescribed. Continues on page 2

2 Volume 15, Number 1

PCR results can be transiently negative during anti-

biotic treatment even though infection persists.

Acute infections generally have higher DNA copies

in samples than chronic infections, because in

chronic infections the immune response has

attenuated the organism. Thus, the optimal

PCR assay sample is usually one collected

during the acute phase prior to antimicrobial

treatment.

While many commercial laboratories offer

nucleic acid amplifi cation assays, they may

not be standardized. In addition, some

labs offer little external quality control. For

example, samples from cats with and with-

out FIV infection were sent to four different

laboratories offering FIV PCR assay.1 While

the lab with the best performance was right

on 90 percent of the samples, two fell below

60 percent. For this reason, CSU VDL main-

tains stringent quality control and standard-

ized protocols for all PCR assays.

While PCR assays are very sensitive, the PPV

of many assays can be very low. For example,

because the technique detects DNA or RNA of

both live and dead organisms, positive test results

may be achieved even if the infection has been

controlled. When the organism being tested for

commonly infects the background population

of healthy pets, interpretation of results for a

single animal can be diffi cult. For example,

healthy cats commonly carry FHV-1, and

modifi ed live vaccine strains colonize cats.

Thus, although PCR is a sensitive way

to document FHV-1 infection, its FHV-1

PPV is actually very low.2-4 In one study

of cats with and without conjunctivitis,2

more FHV-1 positive tests were detected

in the healthy control group than the

group with conjunctivitis. The same prob-

lems exist with feline calicivirus. The

available RT-PCR assays can’t distinguish

between regular calicivirus, vaccine strains

of calicivirus and virulent systemic cali-

civirus. This situation may occur with

herpesvirus and other viral diseases in

other species, such as cattle and dogs.

Consider these realities when inter-

preting test results.

Real-time PCR, or fl uorogenic

PCR, can be more sensitive than con-

ventional PCR for some organisms.

In addition, this assay can be used to

determine the amount of microbial DNA in a sample.

When used quantitatively, this technique can be used to

monitor response to drug treatment.5 It is possible the

DNA or RNA load in a sample will correlate to the pres-

ence of disease for some agents.

A VALUABLE TOOL...WHEN APPLIED RIGHTDepending on the lab, PCR assays can be offered as indi-

vidual tests or in panels. CSU VDL generally offers indi-

vidual assays that

can be coupled as

indicated by the

individual case. For

example, it has been

documented that

haemoplasmas, but

not Bartonella spp.

or Rickettsia spp.,

are associated with

hemolytic anemia

in cats. Thus, for cats with hemolytic anemia, only hemo-

plasma PCR is needed. In contrast, all three genera are

associated with fever of unknown origin, and the assays

can be selected alone or in appropriate combinations.

It’s important practitioners carefully assess the pre-

dictive values of available PCR and the expertise and

reliability of the lab. New PCR assays are being devel-

oped almost daily. At CSU VDL, each PCR assay is opti-

mized prior to being offered commercially, to ensure

quality control is stringently maintained. In addition, an

appropriate specialist in bacteriology, virology or para-

sitology oversees the performance of each assay and is

available for consultation. ▲

REFERENCES1 Crawford PC, Slater MR, Levy JK. Accuracy of polymerase chain

reaction assays for diagnosis of feline immunodefi ciency virus infection in cats. J Am Vet Med Assoc. 2005 May 1;226(9):1503-7.

2 Burgesser KM, et al. Comparison of PCR, virus isolation, and indirect fl uorescent antibody staining in the detection of naturally occurring feline herpesvirus infections. J Vet Diagn Invest. 1999 Mar;11(2):122-6.

3 Low HC, et al. Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis. Am J Vet Res. 2007 Jun;68(6):643-8.

4 Maggs DJ, Clarke HE. Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines. Am J Vet Res. 2005 Sep;66(9):1550-5.

5 Tasker S, et al. Effect of chronic feline immunodefi ciency infection, and effi cacy of marbofl oxacin treatment, on ‘Candidatus Mycoplasma haemominutum’ infection. Microbes Infect. 2006 Mar;8(3):653-61.

6 Clark M, et al. A commercially available Giardia spp. antigen assay detects the assemblages isolated from dogs. Proceedings of the ACVIM Forum, 2008.

PCR DETECTION OF INFECTIOUS DISEASES Continued from page 1

‘ At CSU VDL, an appropriate faculty specialist in bacteriology, virology or parasitology oversees the performance of each assay and is available for consultation.’

Spring/Summer 2010 3 LABLINESInnovative PCR Applications

Help target IMA case managementMeasuring antibodies against

erythrocytes has classically

relied upon spherocyte estimation,

presence of agglutination in saline or

direct Coomb’s test. However, eryth-

rocyte antibodies can also develop

during infection by blood borne

agents, notably Babesia spp., Barton-

ella spp., Ehrlichia spp., Mycoplasma

haemocanis (previously Haemobar-

tonella canis) and M. hematopar-

vum. Because those classic tests are

only semi-quantitative, they haven’t

proved accurate in distinguishing

primary immune-mediated anemia

(IMA) from hemolytic anemia

induced by infectious agents.

Flow cytometry can be used to

quantitatively determine the percent-

age of erythrocytes that have IgM or

IgG antibodies bound to the surface.

Experiments performed over the last

several years at Colorado State Uni-

versity have determined:

■ The assay is accurate for up to

72 hours after the sample is col-

lected. Shipping is no longer a

problem.

■ Dogs with 0 percent IgM and IgG

binding are unlikely to have IMA.

■ Dogs with up to 10 percent

IgM or IgG binding can have

either IMA or infectious hemo-

lytic anemia.

■ Dogs with greater than 10 percent

binding are likely to have IMA.

Flow cytometry used on acutely

anemic dogs sampled before treat-

ment, combined with appropriate

PCR assays to amplify species- spe-

cifi c DNA, offers clinics high positive

and negative predictive values. The

protocol can be used to help deter-

mine whether to emphasize immune

suppression or anti-microbial ther-

apy in case management. ▲

Michael R. Lappin, DVM, PhD, DACVIM,CSU VDL Endocrinology and Special Serology Lab Supervisor

Babesia spp. Neither B. canis nor B. gib-

soni infections are common in Colorado

dogs. However, PCR for these agents are

indicated if the dog with hemolytic anemia

is a greyhound (B. canis), a pitbull or

pitbull cross (B. gibsoni), has been bitten

by a pitbull terrier (B. gibsoni), or has a

recent history of Rhipicephalus ticks (B.

canis).

Bartonella spp. Recently, B. henselae and

B. vinsonii have been detected on dogs in

Colorado and Wyoming. These agents are

uncommon causes of hemolytic anemia

but have been associated with cases

presumed to have primary immune medi-

ated disease that then fail to respond to

immune suppression. All dogs with sus-

pected infectious endocarditis should be

screened with this PCR.

Ehrlichia group. Ehrlichia spp.,

Anaplasma spp., and Neorickettsia are

uncommon causes of hemolytic anemia.

However, if a dog is seropositive or has

recent known exposure to Rhipicephalus

ticks, the assay may be indicated. These

agents are more commonly associated

with thrombocytopenia. However, in acute

cases with fever, epistaxis, polyarthritis

or thrombocytopenia, Ehrlichia group PCR

assay is indicated, particularly if antibody

tests are negative.

Hemoplasmas. All dogs suspected to

have immune or infectious hemolytic

anemia should be screened for

Mycoplasma haemocanis and M. hae-

matoparvum DNA by PCR assay. These

agents are commonly negative by cytol-

ogy, and there is no antibody assay.

PCR ASSAY SELECTION RECOMMENDATIONS

SAMPLE

■ 1 ml of EDTA bloodfor fl ow alone

■ 2 ml if combiningwith the PCR assays

ShippingDeliver on a cold pack to arrive within 48 hours of collection and before noon Fridays. Samples that arrive after noon Friday will not be assayed by fl ow cytometry, but the PCR assays will still be completed.

ScheduleMonday through Friday with results returned the next day

Prices■ Flow cytometry alone $50

Flow cytometry...■ Plus one PCR assay $75■ Plus two PCR assays $100■ Plus three PCR assays $125■ Plus four PCR assays $150

CANINE

4 Volume 15, Number 1

We are often asked by a referring DVM or an

owner: “Hi Doc. What killed my puppy (or

puppies) and what is the best diagnostic protocol?”

Among the numerous potential causes of stillbirth

or post-natal death, including toxic milk syndrome,

mycoplasmosis, neonatal septicemias and

canine brucellosis, an investigative necropsy

should include investigation for canine

herpes virus.

CHV, also known as “fading puppy syn-

drome,” is a viral infection of the adult

reproductive and respiratory tracts. While

adult dogs with latent infections may remain

asymptomatic for a long period of time, they

usually pass the virus in their vaginal secre-

tions or as a droplet during sneezing. Pup-

pies contracting the infection during the fi rst

three weeks of life are the most vulnerable.

An entire litter may succumb to the infection within

a 24-hour period. Death may occur abruptly without

any premonitory signs.

Diagnosis of CHV infection in puppies is usually

made at necropsy. Characteristic gross lesions are

evident in the kidneys as petechial to ecchymotic

hemorrhage and necrosis producing “speckled” kid-

neys. Also, necrosis and

hemorrhage can occur

in the liver and lungs.

MOLECULAR DIAGNOSIS CAN HELPBecause the causative CHV virus is quite labile and

is shed only occasionally and in small amounts, rou-

tine rapid diagnosis of CHV infections by conventional

viral culture or serology is becoming more diffi cult

and unreliable. Antibody levels are often very low and

sometimes undetectable.

Polymerase chain reaction offers a highly sensitive,

rapid and specifi c method to identify the virus in dead

puppies or clinical samples from a symptomatic or

asymptomatic bitch or stud. Genital, nasopharyngeal,

conjunctival or throat swab can be suffi cient for diag-

nosis. Also a small amount of blood, tracheal wash, and

frozen or fresh tissue can prove recent exposure to the

virus.

CSU VDL offers a reasonably priced molecular diag-

nosis to help you quickly evaluate whether the rest of

the litter is at risk of CHV. ▲

Canine Virology

PCR to refi ne differential diagnosisTawfi k Aboellail, BVSc, MVSc, PhD, DACVP,CSU VDL Pathologist

We often think of using the PCR technique to

confi rm that a particular organism is present.

But what about using it to determine that an organism

is not?

Such was the case with a wolf shedding taeniid

tapeworm eggs. This animal had been rescued

from a property in the upper midwest

and brought to Colorado. Fecal fl otation

indicated the animal was shedding taeniid

tapeworm eggs. These types of eggs are usu-

ally thought to be a species of the genus

Taenia, a common tapeworm of canids throughout

the United States. However, this particular animal

came from an area in which wolves harbor both

Taenia and another tapeworm: Echinococcus mul-

tilocularis. While most species of Taenia found in

canids are not zoonotic, E. multilocularis is. Plus, this

parasite is not known to exist in Colorado. Trans-

porting animals into new areas or reintroducing

them into areas where they once thrived is one factor

responsible for the spread of many parasites. Thus,

it was imperative that the true identity of these tape-

worm eggs be determined.

Eggs of these species are quite diffi cult to tell

apart based just on morphology. Therefore, CSU VDL

employed molecular techniques. After extracting the

DNA, amplifying it through PCR and then sequenc-

ing the resulting amplicons, it could be determined

that these eggs were not E. multilocularis, but were

Taenia krabbei, a common species of tapeworm found

in wolves and other wild canids. Transporting this

animal from an E. mulilocularis-endemic area into a

non-endemic area does not appear to have spread this

particular parasite. ▲

Molecular Parasitology

Need the ability to rule something out?Lora R. Ballweber, DVM, MS,CSU VDL Parasitology Section Head

© G

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ey Ku

chera

| Dr

eams

time.c

om

“Turkey-egg” appearance of kidneys is due to hemorrhage and tissue necrosis. Diagno-sis was confi rmed in less than 24 hours by PCR.

A carrier or not? CSU VDL used PCR to rule out the presence of a potentially zoonotic tapeworm species in a transplanted wolf, ensuring the animal wasn’t intro-ducing the parasite into Colorado.

FOCUS ON PCR

Spring/Summer 2010 5 LABLINESCanine Oncology Innovations

New canine cutaneous mast cell tumor profi le helps guide therapy In 1999, a group of researchers at Harvard Medical

School led by a veterinary oncologist discovered an

important clue to the pathogenesis of mast cell tumors

in dogs.1 Eventually, the discovery of that mutation and

its effects on mast cells led to the development of a

new drug, Palladia, a tyrosine kinase inhibitor, specifi -

cally targeted to treating canine mast cell tumors. Test-

ing now available through the Diagnostic Laboratory,

as a component of the new Canine Cutaneous Mast

Cell Tumor Profi le, can identify this mutation and help

guide therapeutic decisions.

IDENTIFYING GROWTH DRIVERSMast cell growth is driven in part by the binding

of a growth factor (stem cell factor) to a tyrosine

kinase receptor (c-kit) expressed on the surface of

mast cells. When the

growth factor binds, the

intracellular portion of

c-kit becomes phos-

phorylated, initiating a signaling cascade that eventu-

ally leads to mast cell division. The Harvard researchers

found about 20 percent of canine mast cell tumors car-

ried a mutation in the c-kit gene called an internal

tandem duplication, or ITC; part of the gene is dupli-

cated. The biological consequence of this duplication

was a change in the protein structure of c-kit, which

causes it to be permanently phosphorylated and consti-

tuitively active, even when the growth factor is not pres-

ent. The unchecked growth we see in mast cell tumors

results.

The new tyrosine kinsase inhibitor Palladia inhibits

c-kit phosphorylation, and therefore effectively inhib-

its mast cell growth. Recent clinical trials have dem-

onstrated the effectiveness of Palladia as a therapy for

mast cell tumors that have recurred after excision.2 Pal-

ladia was most effective in patients whose tumors car-

ried the c-kit mutation, at 60 percent to 70 percent

effective, compared to patients whose tumors did not

carry the mutation, at only 30 percent to 40 percent

response. Detecting the c-kit mutation can help guide

a decision to use Palladia over other chemotherapeutic

agents such as vinblastine.

In addition to the evaluation for a c-kit gene

mutation, the new canine cutaneous mast cell tumor

(MCT) profi le also assesses the proliferation index of

the tumor via a combination of mitotic index and

Ki67 expression, a marker

expressed during all

stages of active cell

division. Plus, it

evaluates expres-

sion and localiza-

tion of the c-kit

receptor/protein.

P r o l i f e r a t i o n

index, both through

mitotic index3,4 and

Ki67 expression,5 has

been demonstrated to

inversely correlate with local recur-

rence, incidence of distant metastases and overall

survival. Additionally, localization of the c-kit

protein — specifi cally, aberrant cytoplasmic expres-

sion) — has also been demonstrated to correlate with

local recurrence and overall survival times, where Type

II and III localization (focal and diffuse cytoplasmic,

respectively) are associated with increased local recur-

rence and decreased survival times.6

PUTTING THE ASSAY TO WORKCSU VDL’s DNA assay can be run on air-dried stained

or unstained aspirates, as well as on biopsy formalin

fi xed tissues; however at least 10 percent of the cells

in the aspirate or biopsy must be mast cells in order

for the mutation to be detected. The proliferative index

and IHC stain for Kit localization are carried out on

biopsy samples. All tests can be run independent of the

MCT profi le, but the combined information obtained

via the complete profi le provides optimal prognostic

and therapeutic guidance.

For information on this new MCT profi le, includ-

ing how to request one and when the test is run,

visit the website dlab.colostate.edu/webdocs/services/

MCTProfi leInformation.pdf or call (970) 297-1281.

Anne Avery, at anne.avery@colostate.edu, can answer

questions on the c-kit mutation assay, and Brad Charles,

at (970) 297-4087 can provide more information about

the mast cell tumor profi le. ▲

1. London CA, Galli SJ, Yuuki T, et al. Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Exp Hematol. 1999 Apr;27(4):689-97.

2. London CA, Malpas PB, Wood-Follis SL, et al. Multi-center, placebo-controlled, double-blind, randomized study of oral toceranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, for the treatment of dogs with recurrent (either local or distant) mast cell tumor following surgical excision. Clin Cancer Res. 2009 Jun 1;15(11):3856-65.

3. Romansik EM, Reilly CM, Kass PH, Moore PF, London CA. Mitotic index is predictive for survival for canine cutaneous mast cell tumors. Vet Pathol. 2007 May;44(3):335-41.

4. Elston L, Sueiro FA, Cavalcanti JN, Metze. The importance of the mitotic index as a prognostic factor for canine cutaneous mast cell tumors - a validation study. Vet Pathol. 2009 Mar;46(2):362-364.

5. Webster JD, Yuzbasiyan-Gurkan V, Miller RA, et al. Cellular proliferation in canine cutaneous mast cell tumors: associations with c-KIT and its role in prognostication. Vet Pathol. 2007 May;44(3):298-308.

6. Webster JD, Yuzbasiyan-Gurkan V, Thamm DH, et al., Evaluation of prognostic markers for canine mast cell tumors treated with vinblastine and prednisone. BMC Vet Res. 2008 Aug 13;4:32.

Anne Avery, DVM,PhD, CSU Microbiology, Immunology and Pathology Assistant Professor; EJ Ehrhart, DVM, PhD,DACVP, CSU VDL Pathologist; and Debra Kamstock, PhD,DVM,DACVP, CSU VDL Pathologist

FOCUS ON PCR

Mast cell growth process

Normalc-kit

Mutatedc-kit

Stem cellfactor

binds to c-kitC-kit is

phosphorylated

Mast cells undergo limited

division

Mast cellsdivide

continuously

Mutated c-kitis always

phosphorylated

6 Volume 15, Number 1

As a member of the National Animal Health Labo-

ratory Network, CSU VDL is approved to perform

infl uenza real-time reverse transcription PCR. Five assays

are available to NAHLN labs, and CSU VDL performs all

fi ve: infl uenza A viruses (matrix gene), H5 avian infl u-

enza viruses, H7 avian infl uenza viruses and the pan-

demic H1N1 infl uenza virus (matrix gene

assay and N1 assay). All positive samples are

sent to the National Veterinary Services Lab

in Ames, Iowa, for confi rmatory testing.

In 2004, CSU VDL and the Colorado

Department of Agriculture started the Col-

orado Avian Disease Surveillance Program.

Funded by USDA and housed at VDL,

the program provides free avian infl uenza

surveillance testing for commercial fl ocks,

backyard fl ocks and live bird markets. The

program also provides free disease inves-

tigation services for backyard fl ock owners experienc-

ing disease and mortality. In addition, the CSU VDL has

also participated in the national USDA-Wildlife Services

wild bird avian infl uenza surveillance program.

Since 2004, more than 30,000 Colorado wild and

domestic bird samples have been tested for avian infl u-

enza virus. We have detected low pathogenic avian infl u-

enza viruses, primarily in wild bird samples. No highly

pathogenic avian infl uenza viruses have been identifi ed.

In 2009, with the emergence of pandemic H1N1

infl uenza virus, the NAHLN released a modifi ed infl u-

enza A matrix real-time PCR protocol, as well as a

real-time PCR protocol targeting the 2009 N1 subtype.

While the primary use for these assays is to test swine,

we were granted permission to use them to screen

companion animals.

To date, we have tested 14 samples from swine, cats

and dogs. All swine and canine samples have tested

negative. CSU VDL diagnosed two cases of H1N1 pan-

demic infl uenza virus in domestic cats in November

2009. These cases were some of the fi rst cases diag-

nosed in companion animals. Both cats presented to

veterinarians with respiratory abnormalities and expe-

rienced a prolonged period of illness. Information on

these two cases is in preparation for publication. ▲

Guardians of Public Health

Real-time infl uenza PCR testingKristy Pabilonia, DVM, DACVM, CSU VDL Avian Diagnostics and BSL3 Operations Section Head; and Christina Weller, CSU VDL Avian Diagnostics and BSL3 Operations Microbiologist

A young adult, mixed-breed bitch whelped a litter of

11 puppies while in a rescue facility. Several became

ill at 5 weeks old. One died and was diagnosed with infec-

tious canine hepatitis (ICH). Two unrelated 4-month-

old puppies were exposed.

Urine samples were sub-

mitted from nine remaining,

8-week-old puppies, the dam

and the two exposed dogs for PCR canine adenovi-

rus-1 (CAV-1) testing. All nine puppy sample were

positive for CAV-1 by PCR, while the dam and exposed

puppy samples were negative.

CAV-1 is the etiologic agent for ICH. The clinical

signs in the acute phase of ICH — fever, anorexia, leth-

argy, vomiting and diarrhea — are non-specifi c and may

be mistaken for parvovirus-1 or distemper. Frequently,

ICH is suspected only when some dogs develop corneal

edema or “blue-eye” in the subacute phase, at three to

four weeks after infection. The disease is more severe in

younger dogs, and peracute death may occur in puppies.

The virus targets the lymphoid tissues, including the

tonsils and Peyer’s patches; the liver, causing hepatitis; and

the kidneys. Although the immune response clears the

virus in 10 to 14 days, the virus persists in the kidneys of

survivors and is shed in urine for weeks to months.

In addition to dogs, CAV-1 infects foxes, coyotes,

wolves, raccoons, ferrets, skunks and bears. Immuni-

zation with CAV-2 containing modifi ed live viral vac-

cines has been effective in preventing ICH in domestic

dogs. While uncommon in vaccinated populations,

ICH occasionally crops up in unvaccinated dogs in

rescue and humane shelters. In suspect cases, whole

EDTA blood may be submitted for PCR. Tissues includ-

ing liver, spleen, kidney and lymph nodes are suitable

post-mortem samples. Urine samples should be tested

for shedding. The cost of the PCR is $35. ▲

Case Study

Infectious hepatitis: Forgotten, not goneJeanette Bishop, MS, CSU VDL Molecular Diagnostics Research Associate, and Hana Van Campen, DVM,PhD,DACVM, CSU VDL Virology Section Head

Contact Kristy Pabilonia at (970) 297-4109 if you have questions about infl uenza testing

FOCUS ON PCR

CSU VDL’s cooperative Avian Disease Surveillance Program helps monitor the growing urban poultry movement for early warnings of emergent pandemic infl uenza.

Spring/Summer 2010 7 LABLINES

Tritrichomonas foetus Positives

Tested

by pooled PCR

(n = 27,856)

Tested by individual PCR

(n = 8,675)

1.0%(n = 288)

0.9%(n = 79)

Lab Update

TREKing Johne’s for faster resultsCSU VDL’s Bacteriology Section offers a more

rapid test for the detection of Mycobacterium

avium subspecies paratuberculosis. Instead of using

the old method of culturing the sample onto solid

media and checking for

growth over a period of

112 days, bacteriology

now performs a liquid

culture using the TREK para-JEM ESP Culture System

II, which checks for bacterial growth over a period of

45 days.

The TREK system works by continuously monitor-

ing the liquid culture for bacterial growth, notifying

the technician of positive growth with a light. Just

because there is growth does not necessarily mean

the sample contains M. avium paratuberculosis. The

sample is put through a decontamination process,

but some organisms, such as other mycobacteria spe-

cies, are hardy enough to survive the process and will

grow in the liquid culture, giving a false positive. To

ensure no false positives are reported, we perform a

real-time PCR detecting M. avium paratuberculosi-

sis after growth in the liquid culture. If the sample

is PCR-positive, the client will receive an M. avium

paratuberculosisis-positive report. If the sample is

PCR-negative, the liquid culture will be put back

onto the TREK system for the remainder of the 45

days. The PCR then will be performed again on day

45 to ensure the sample is still negative. ▲

Mike Russell, CSU VDL Microbiologist, and Doreene Hyatt, PhD, CSU VDL Bacteriology Section Head

CSU ROCKY FORD DEMONSTRATES POOLED TRICH SAMPLE RELIABILITY

From Jan. 1, 2007, to May 5, 2010, the CSU VDL at Rocky Ford has tested 27,856 bulls by pooled PCR for the presence of T. foetus and found 288 positive individuals. During the same period, the lab has tested 8,675 by individual PCR and found 79 positives. The number of positive by pooling is 1 percent, while the number by individual PCR it is 0.9 percent. Statistical analysis of the test results show no signifi cant statistical difference in the number of positives detected by pools versus the number of positives detected by individual PCR. Pooling can increase the cost-effectiveness of Trich testing without compromising diagnostic reliability.

VDL NOW NVSL-APPROVEDFOR JOHNE’S DIRECT PCR DETECTION

VDL is pleased to inform you it has passed the individual 2010 Johne’s Profi ciency Panel using direct PCR. This update will permit us to conduct offi cial testing for USDA beginning immediately through the end of 2011. The real-time PCR detection of M. avium paratuberculosisis directly from a fecal sample will allow clients to receive a result many times on the same day we receive the sample.We have also passed our profi ciency test for liquid culture, solid culture and serum ELISA for Johne’s disease and are approved by USDA through the end of 2010.

BOVINE

Real-time PCR testing for T. foetus is now avail-able at the Fort Collins lab. Quicker results with no change in sample col-lection or increase in price compared to con-ventional testing. Email dlab@colostate.edu or call (970) 297-1281for details.

ATTENTION: NEW LIMS!

Starting July 1 we switched to a new laboratory information management system (LIMS), call STARLIMS. You will see new and improved formats for your results. Please call with questions and comments. Please be patient with as we go through this transition.

8 Volume 15, Number 1

USDA has now authorized CSU VDL to perform the

cELISA tests for Theileria equi and Babesia caballi

for the intra- and interstate movement of equids not dis-

playing clinical signs of piroplasmosis. Tests are

run each Tuesday and Friday. Submit 1 ml of

serum and be sure to include horse’s iden-

tifi cation. Cost is $16 per sample.

As was reported in April, a horse in Col-

orado tested positive for equine piroplas-

mosis. Although it showed no clinical signs of

equine piroplasmosis, the horse tested positive to Theile-

ria equi on the cELISA test. The serum sample had been

submitted by a Colorado private practitioner. The posi-

tive horse has since been euthanized.

Arapahoe Park Racetrack in Aurora instituted EP test-

ing requirements for all horses entering the track facil-

ities — the reason the EP-positive sample was

submitted. Horses must be T. equi- and Babe-

sia caballi-negative within 30 days of admission to Arap-

ahoe Park. The piroplasmosis test-positive horse was in

a stable with about twenty horses. None of those horses

are considered positive at this time. Further testing will

be done in approximately 30 days from the initial tests. If

they are negative, the quarantine will be released.

Equine piroplasmosis is a reportable disease and

leads to regulatory consequences. A quarantine and

subsequent disease-control plan for any test-positive

horses is determined by the State Veterinarian of Col-

orado, USDA-APHIS-VS, along with input from the

owners. Currently, there is no vaccine or approved

treatment for EP in the United States.

USDA-APHIS-VS’ Equine Piroplasmosis Info Sheet,

written in English and Spanish, provides further infor-

mation for you and your clients. You can access it

on the Web at www.aphis.usda.gov/animal_health/

animal_diseases/piroplasmosis/index.shtml. ▲

Reportable Disease Update

Colorado horse tests EP-positive

A sample of lung from a 4-month

old dairy calf was submitted

to the diagnostic laboratory for

histopathology and FA

tests. Entire lobules were

positively stained for

bovine respiratory syn-

cytial virus by FA test. The pattern of

BRSV staining is unusual compared

to that observed in bovine pneu-

monias. Histologically, we observed

a severe interstitial pneumonia with

multinucleated syncycitial cells.

Lung tissue was also assayed for

BRSV using a reverse transcriptase

PCR test. It was strongly positive

for BRSV genomic RNA.

Due to the histologic fi ndings and

the extensive FA staining in the lung,

vaccination history of the calf was

obtained. Calves were inoculated by

intranasal route with a modifi ed

live BRSV vaccine labeled for intra-

muscular administration. Six calves

became ill, fi ve responded to treat-

ment and one died.

Attenuated live viral IM vaccines

may result in disease when admin-

istered by the intranasal route.

Unforeseen environmental factors,

such as immune status, and other

infectious agents may tip the bal-

ance of health in calves inoculated

intranasally with BRSV. Recent

reports suggest intranasal vaccina-

tion for BRSV in young calves may

not fully protect against subsequent

viral challenge due to the presence

of maternal antibodies.1 ▲

Bovine Virology Case Study

An unusual case of BRSV in a calfChristina Gates, CSU VDL Microbiologist; Jeanette Bishop, MS, CSU VDL Molecular Diagnostics Research Associate; Barb Powers, DVM,PhD,DACVP, CSU VDL Director; and Hana Van Campen, DVM,PhD,DACVM, CSU VDL Virology Section Head

Questions? Callthe State Veterinarian’s Offi ce at (303) 239-4161

FOCUS ON PCR

1 Ellis JA, Gow SP, Goji N. Response to experimentally induced infection with bovine respiratory syncytial virus following intranasal vaccination of seropositive and seronegative calves. J Am Vet Med Assoc. 2010 May 1;236(9):991-9.

1-100 bp ladder

Calf lung

1:10 dilution of calf lung

Negative extraction control

BRSV positive amplifi cation control

Negative amplifi cation control

Spring/Summer 2010 9 LABLINESGaurdians of Public Health

Meeting the challenge of Q feverQ fever is a zoonotic disease that results from infec-

tion with the organism Coxiella burnetii. This

obligate intracellular Gram-negative bacterium is dis-

tributed worldwide. Cattle, sheep and goats are the pri-

mary reservoir of the organism. The organism is shed

in milk, urine, feces, amniotic fl uid, birth fl uids and

placenta of infected animals. The organism can sur-

vive for prolonged periods in the environment and

exposure to only a few organisms can result in infec-

tion. Animal infections are often asymptomatic. The

primary clinical manifestations observed in animal

populations are aborted fetuses and birth of weak or

nonviable neonates.

ZOONOTIC POTENTIALMost human cases results from contact with cattle,

sheep and goats, especially during parturition. Human

cases can also potentially result from consumption

of unpasteurized milk. Infection is acquired through

inhalation or ingestion of the organism. Companion

animals are also susceptible and have been documented

as the source of human infections. Q fever can result

in acute and chronic cases in humans. In acute cases,

symptoms include fever, vomiting, diarrhea, atypical

pneumonia, headache, hepatitis, skin rash, myalgia and

general malaise. In chronic cases, the disease persists

for six months or longer and can result in severe dis-

ease and endocarditis. Infection during pregnancy may

result in abortion, fetal death, premature delivery and

death. Q fever is a reportable disease, but because

approximately 60 percent of human cases are asymp-

tomatic, it is often under-reported.

In 2009, the Netherlands diagnosed more than 2,300

human cases and a large scale effort was instituted to

control prevalence of the organism in small ruminants,

with the hope that this would decrease incidence of

human infection. Control measures include culling of

tens of thousands of pregnant goats on affected farms

and mass vaccination. The impact of these actions con-

tinues to be evaluated by the scientifi c community and

it is hoped will provide insight on the best way to

handle this type of situation.

DIAGNOSTIC CHALLENGESBecause infected animals do not always shed organ-

isms, diagnostic testing for Q fever can be diffi cult.

A negative PCR result does not guarantee that the

animal or population is not infected. Repeated sam-

pling and testing should be used before an animal or

farm is determined to be Q-fever negative. Antibody

testing should be interpreted with care. Current serol-

ogy techniques lack desired sensitivity and specifi city,

and seropositivity can be diffi cult to interpret as it is

diffi cult to differentiate between acute infections and

past exposure.

CSU VDL MONITORING UPDATESince we began offering a real-time PCR assay for C. bur-

netii, the number of submissions has grown from

21 in 2007 to more than

300 in 2009. Submissions

have been received from

bovine, camelid, canine,

caprine and ovine species.

We receive samples for diag-

nostic testing for animals

with clinical manifestations

of Q fever and from animals

on premises with Q-fever

positive owners. In addition, we receive submissions for

screening research animals and for export testing.

As the overall number of submissions has grown,

the prevalence of positive samples has decreased. In

2007, 8.7 percent of the total samples tested positive;

however, this amounted to only two samples out of a

total of 21 tested. In 2008, 2.6 percent of samples tested

positive, with no confi rmed positives in 2009.

We have recently started offer-

ing the CDC Laboratory Response

Network’s real-time PCR assay for

Q Fever. This assay incorporates

three primer/probe sets, each of which targets a different

sequence of the C. burnetii genome. This PCR will allow

us to provide a higher level of diagnostic sensitivity.

However, the increase in the number of target sequences

from one to three means that there is an increased test

cost. The cost of this test is $60 per sample. C. burnetii

organisms can be found in the lungs, spleen and liver, as

well as milk and blood, so these samples are best for test-

ing in non-abortion cases. In abortion cases, placenta

is the best sample for diagnostic testing. Tissues, swabs

and milk samples from multiple animals may be pooled

for testing, but this may decrease overall sensitivity. In

order to minimize exposure of our laboratory personnel

to this agent, we perform this test in our new biosafety

level 3 laboratory. ▲

Kristy Pabilonia, DVM, DACVM, CSU VDL Avian Diagnostics and BSL3 Section Head; and Christina Weller, CSU VDL Microbiologist

Contact Kristy Pabiloniaat (970) 297-4109 if you have questions about Q fever testing

Q Fever Positives

8.7%

2.6%

0%20092007 2008

FOCUS ON PCR

10 Volume 15, Number 1

Spraker TR, et al. Detection of the Abnormal Isoform of the Prion Protein Associatedwith Chronic Wasting Disease in the Optic Path-ways of the Brain and Retina of Rocky MountainElk. Vet Pathol. 2010 May;47(3):536-46.CSU VDL Pathologist Terry Spraker examined the eyes

and nuclei of the visual pathways in the brains of 30

Rocky Mountain elk (Cervus elaphus nelsoni) for the

presence of the abnormal isoform of the prion protein

associated with chronic wasting disease, PrP.CWD The

research team found the prion in the retina of 89 per-

cent of the elk showing an obex score higher than 7

(ranging from a disease-free 0 to a terminal 10), com-

pared to no detection in disease-free elk or those with

an obex below 6. The elk with an obex score of less than

6 had no evidence of PrPCWD immunoreactivity in

the optic nerve or retina but did have PrPCWD immu-

noreactivity in neural regions of the visual pathway

in the brain. The data demonstrate detectable PrPCWD

immunoreactivity fi rst accumulates in the visual path-

ways of the brain, before the retina.

McCoy AM, Hackett ES, Callan RJ, Powers BE. Alimentary-associated carcinomas in fi ve Viet-namese potbellied pigs. J Am Vet Med Assoc. 2009 Dec 1;235(11):1336-41.VDL Director Barb Powers assisted a CSU Department

of Clinical Sciences team in documenting the cases of

fi ve geriatric Vietnamese potbellied pigs presenting to the

CSU Veterinary Hospital with complaints of abdominal

distress that had not responded to medical treatment,

in the case of four, and a draining tract of

the cranial abdomen of unknown duration, in

the fi fth. Although neoplasia of gastrointes-

tinal or hepatobiliary origin in pigs is infre-

quently reported — and never yet reported

in Vietnamese potbellied pigs — the study

team tracked the cases of the fi ve pigs with

primary intestinal tumors (in three), gastric

carcinoma (in one), and undifferentiated

carcinoma of billiary origin (in one).

Neoplasia is generally considered rare

in pigs, but it shouldn’t be overlooked

in the differential diagnosis of generalized

abdominal distress in middle-aged and older

potbellied pigs. As was evident in the report,

tumors can potentially arise from multiple

components of the gastrointestinal and hepato-

biliary tracts. If neoplasia is suspected, tumor

excision may provide a good outcome: All three pigs that

were treated surgically survived at least nine months

after surgery with good quality of life.

Ballweber LR. Taenia crassiceps subcutaneous cysticercosis in an adult dog. Vet Rec. 2009 Dec 5;165(23):693-4. Lora Ballweber, CSU VDL Parasitology Section head,

details the case of an eight-year-old adult blue heeler

cross presented to the referring emergency clinic with an

abscess on the left hindlimb. A large, fl uctuant swelling

was present on the medial left thigh; cytology revealed

numerous degenerative neutrophils and macrophages

with no other abnormalities. Ovoid parasites containing

clear watery fl uid with a single invaginated scolex identi-

fi ed upon diagnostics were identifi ed as T. crassiceps cys-

tice. T. crassiceps cysticercosis in dogs as been reported

only six times previously in Europe and only once in

the United States. T. crassiceps not only poses a fatal

threat for dogs and cats, it is a clear zoonotic risk as well.

Ballweber reminds veterinarians they should remember

dogs are defi nitive hosts for multiple species of Taenia,

including crassiceps. Comprehensive pet parasite control

should continue to be a priority.

Ballweber LR, et al. Giardiasis in dogs and cats: update on epidemiology and public healthsignifi cance. Trends in Parasitology 2010 Apr;26(4):180-9.Ballweber updates the status of molecular identifi cation

of the seven genetic assemblages of Giardia duodenalis,

named A to G. Humans are infected with assemblages

A and B, dogs primarily with C and D, and cats with

F. Recently, small numbers of dogs and cats have been

reported to also carry assemblages A-I or B. Although

the role of dogs and cats as a source of human giardia-

sis remains unresolved, the potential role of pets can’t be

conclusively excluded, she cautions. ▲

CSU VDL In Press

A roundup of VDL faculty research

Ballweber documents the budding cysticerci of Taenia crassi-ceps from a swelling on the leg of a dog (left) and a squash mount of an inverted scolex showing typical rostellar hooks.

Spring/Summer 2010 11 LABLINES

Veterinary Community Outreach

External advisory committeeOur External Advisory Committee members vol-

unteer their time to meet with us annually and

assess our progress, as well as provide input to our

future directions. We are grateful for their time and

advice, and hope they feel they are an integral part

of the laboratory.

WIN A $50 CREDITON LAB WORK

Take a short fi ve-minute survey to tell us how to improve LabLines and we’ll enter you in a drawing to win a $50 credit on CSU VDL services.

To begin the survey, use your Internet browser to go to:FoodChainCommunications.com/Lablines

Some restrictions apply. See website for details.

NEW DIAGNOSTIC SCREENS

CSU VDL has introduced new diagnostic respiratory, diarrhea and abortion screens. These species-specifi c screens combine tests for the most commonly diagnosed viral, bacterial, parasitic and toxicological agents of respiratory, diarrhea and abortion cases. In most cases, histology is also included.The costs of these panels vary depending

on the species and type of panel, but all are offered at a discount from the cost of running all the included tests individually.Look for these new tests on Page 2 of the General Submission Form.Detailed descriptions of these panels can be viewed at dlab.colostate.edu. Scroll down to the In the News section and click on Panels and Screens.

■ Dr. Joan Bowen, Small Ruminant

Wellington

■ Mr. Norm Brown, Equine

Wellington

■ Dr. Keith Roehr, State Vet

CO Dept of Agric, Denver

■ Mr. Terry Fankhauser, Executive

Director, CCA

Arvada

■ Dr. Mike Gotchey, Equine

Steamboat Springs

■ Dr. Bob Davies, Wildlife

Colorado Dept of Wildlife

Denver

■ Dr. Marv Hamann, Mixed Practice

Pueblo West

■ Mr. Ed Hansen, Beef Cattle

Livermore

■ Dr. Dean Hendrickson, Director

CSU Veterinary Teaching Hospital

Fort Collins

■ Dr. Ron Kollers, Small Animal

Fort Collins

■ Dr. Elisabeth Lawaczeck

Colo. Dept of Public Health

and Environment

Denver

■ Dr. Larry Mackey, Large Animal

Greeley

■ Dr. Leesa McCue, Mixed Animal

Limon

■ Dr. Del Miles, Beef Cattle

Greeley

■ Dr. Mike Miller, Wildlife

Colorado Dept

of Wildlife

Fort Collins

■ Dr. Roger Perkins

USDA/AVIC/APHIS

Lakewood

■ Mr. Kenny Rogers, CCA

Yuma

■ Dr. Steve Wheeler, Small Animal

Englewood

■ Dr. Deb Young, CSU Extension

Fort Collins

JOE MCDOWELLCraig, Colo., native and University of Northern Colorado grad Joe McDowell joined the VDL in February. Joe will help VDL maintain its commitment to quality assurance and customer service through various lab support functions. Welcome, Joe.

2008■ Barbara E. Powers, DVM

APRIL 2009■ Hana Van Campen

MAY 2009■ James A. Kennedy on behalf

of Ms. Kathy GilbertAUGUST 2009

■ Kristy L. Pabilonia, DVM

NOVEMBER 2009■ Barbara E. Powers, DVM■ Fisher’s Peak Veterinary Clinic on

behalf of William C. Aaroe, DVMDECEMBER 2009

■ Foothills Animal Hospital on behalf of Roger A. Liehr, DVM

■ Constance M. Hvass, PhD, in honor of Dr. Linda Sue Wiest

■ Amanda Hosny, CVT, in honor of Dr. Jenna Burton

■ Ferris Veterinary Services on behalf of Martha A Ferris, DVM

MARCH 2010■ Colorado Cattlemen’s Association

We would like to extend our sincere gratitude to our initial donors to the new CSU VDL Endowment. Thank you for supporting the VDL and our mission. We invite you to join us

on this mission to establish and grow the Endowment Fund. Contribute by visiting us online at www.dlab.colostate.edu and clicking “Support the DLAB online.”

p

BARBARA POWERS,DVM, PHD, DACVP

DIRECTOR

Update from the Director

LABNEWS

Nonprofi t OrganizationUS Postage

PAIDFort Collins, Colorado 80523

Permit Number 19

INSIDE THIS ISSUEINSIDE THIS ISSUEREGULAR COLUMNS

Veterinary Diagnostic LaboratoriesCollege of Veterinary Medicine and Biomedical SciencesFort Collins, CO 80523-1644

FOLLOW OURANNUAL PROGRESS

Check out our 2009 annual report, at

www.dlab.colostate.edu/webdocs/news/

FOCUS ON PCR . . . . . . . . p. 1How PCR improves thedetection and managementof infectious diseases.

IMA MANAGEMENT . . . . . p. 3How CSU VDL’s PCR can helpclinics better manageimmune-mediated anemia.

WHAT KILLED MY PUP? . . p. 4PCR improves differentialdiagnosis of neonatal deaths.

EP TESTING UPDATE . . . . p. 8Equine piroplasmosis appearsin Colorado. What you needto know about testing.

AVIAN FLU . . . . . . . . . . . . p. 6Employing PCR to monitorfor avian infl uenza.

Q FEVER REVIEW . . . . . . . p. 9VDL’s experts detail what weknow about thiszoonotic pathogen.

■ DIAGNOSTIC UPDATE p. 5Canine mast cell profi le.

■ PUBLIC HEALTH p. 6Real-time infl uenza PCR.

■ DIAGNOSTIC UPDATE p. 7Johne’s testing additions.

■ VDL IN PRESS p. 10The latest research roundup from the VDL faculty.

■ MEET THE LAB p. 11

Molecular Diagnostics, specifi cally

Polymerase Chain Reaction (PCR),

is certainly not new to our laboratory or

laboratories across the country. However, in

the recent years, requests for this technol-

ogy have dramatically increased. In general,

PCR technology is accepted to be a very sen-

sitive and specifi c testing procedure. It has

replaced many of the older diagnostic tech-

niques, which were either less sensitive or

took a long time to complete. Does a posi-

tive PCR test always conclusively show the

animal has disease? Likewise, does a nega-

tive PCR test always indicate the animal does not have

disease? This issue of LabLines explores these ques-

tions and demonstrate the proper use of PCR tech-

nology to either make or confi rm a diagnosis of a sus-

pected disease. The lead article by Dr. Mike Lappin also

details some of the issues regarding interpretation of

PCR diagnostics. As always, a practitioner and diagnos-

tician, working as a team, is critical in interpreting the

fi nal result of this — and any — test. That’s why CSU

VDL always includes close oversight and consultation

with an appropriate faculty specialist in bacteriology,

virology or parasitology for each assay

In other news, our 2009 Annual Report

is available on our website or as hard copy

upon request. In January, we met with

our External Advisory Committee, which

as always provided us with guidance on

moving the laboratory forward. Our labo-

ratory, as well as other diagnostic labora-

tories, veterinary clinics and people across

the country have struggled in these eco-

nomic times. But recently, we have seen

an increase in laboratory use, signaling the

economy is truly on its way to recovery.

We hope you all have an enjoyable

summer and enjoy the

warm weather which

has fi nally just arrived

to Colorado after a very

long, cold winter. We look forward to seeing you this

fall at the Colorado Veterinary Medical Association

meeting in Loveland and the Annual American Asso-

ciation of Veterinary Laboratory Diagnosticians meet-

ing in Minnesota.